Small molecule FIEL1 inhibitor in inflammatory and fibrotic lung injury

ABSTRACT

Novel compounds are disclosed along with methods of inhibiting the TGFβ pathway and methods of treating Idiopathic Pulmonary Fibrosis (IPF) using such compounds.

CROSS REFERENCE TO RELATED APPLICATION

This application is a National Stage of International Patent ApplicationNo. PCT/US2016/028614, filed Apr. 21, 2016, which claims priority toU.S. Provisional Patent Application No. 62/151,158, filed Apr. 22, 2015.The contents of these applications are incorporated herein, by referencein their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under NIH grant#HL116472 awarded by the NIH. The government has certain rights in theinvention.

BACKGROUND

An aspect of the present invention relates generally to the field ofcompounds and treatment of diseases associated with an activatedinflammatory pathway or idiopathic pulmonary fibrosis (IPF).

IPF is a progressive fibrotic disease characterized by massiveneovascularization and deposition of extracellular matrix into theinterstitium. IPF is the most common form of interstitial lung diseasewith a prevalence of 50 per 100,000 cases, and it almost exclusivelyaffects patients older than 50. Despite its unknown etiology, thedownstream effectors of IPF are well-characterized. Samples from IPFpatients show increased levels of transforming growth factor beta (TGFβ)across all three isoforms (Annes et al., “Making sense of latent TGFβactivation,” Journal of cell science, 116(Pt 2): 217-224 (2003)). TGFβserves as a critical pro-inflammatory molecule via the induction ofneutrophil chemotaxis, the activation of epithelial to mesenchymaltransition, and the promotion of lung epithelial cell apoptosis (Kage etal., “EMT and interstitial lung disease: a mysterious relationship,”Current opinion in pulmonary medicine, 18(5): 517-523 (2012)). The TGFβsignaling pathway proceeds in part through the mothers againstdecapentaplegic homolog (SMAD) protein family. SMAD proteins regulate avariety of cellular processes, such as differentiation, proliferation,tumorogenesis, and immune responses (Attisano et al., “SMADS astranscriptional co-modulators,” Current opinion in cell biology, 12(2):235-243 (2000); Yingling et al., “Development of TGF-beta signallinginhibitors for cancer therapy,” Nature reviews Drug discovery, 3(12):1011-1022 (2004); Bonniaud et al., “TGF-beta and Smad3 signaling linkinflammation to chronic fibrogenesis,” Journal of immunology, 175(8):5390-5395 (2005)). The SMAD family is comprised of receptor-SMADs(R-SMAD), inhibitor SMADs (I-SMAD), and the common mediator SMAD(co-SMAD) (Deiynck et al., “SMAD-dependent and SMAD-independent pathwaysin TGF-beta family signaling,” Nature, 425(6958): 577-584 (2003)).Briefly, TGFβ signal transduction commences with the phosphorylation ofR-SMADs, often SMAD2 or SMAD3, which form a trimeric structure with theco-SMAD. SMAD4, and translocate to the nucleus to bind to the SMADbinding element (SBE) in the JunB promoter to activate transcription(Jonk et al., “Identification and functional characterization of a SMADbinding element (SBE) in the JunB promoter that acts as a transforminggrowth factor-beta, activin, and bone morphogenetic protein-inducibleenhancer,” The Journal of biological chemistry, 273(33): 21145-21152(1998)). Therefore, TGFβ is a major pro-fibrotic growth factor throughthe downstream SMAD signaling pathway (Wang et al., “Caveolin-1: acritical regulator of lung fibrosis in idiopathic pulmonary fibrosis,”The Journal of experimental medicine, 203(13): 2895-2906 (2006); Heckeret al., “NADPH oxidase-4 mediates myofibroblast activation andfibrogenic responses to lung injury,” Nature medicine, 15(9): 1077-1081(2009)).

PIAS (protein inhibitor of activated STAT) proteins are a family ofproteins that are known to negatively control and regulate genetranscription and inflammatory pathways in cells (Rytinki et al., “PIASproteins: pleiotropic interactors associated with SUMO,” Cell Mol LifeSci, 66(18): 3029-3041 (2009)). There are four characterized PIAS familymembers, PIAS1, PIASx (PIAS2), PIAS3, and PIASy (PIAS4), each withspecificity toward different pathways (Gross et al., “Distinct effectsof PIAS proteins on androgen-mediated gene activation in prostate cancercells,” Oncogene, 20(29): 3880-3887 (2001)). Specifically, PIAS4 hasbeen shown to suppress TGFβ signaling (Long et al., “Repression of SMADtranscriptional activity by PIASy, an inhibitor of activated STAT,”Proceedings of the National Academy of Sciences of the United States ofAmerica, 100(17): 9791-9796 (2003); Imoto et al., “Regulation oftransforming growth factor-beta signaling by protein inhibitor ofactivated STAT, PIASy through Smad3,” The Journal of biologicalchemistry, 278(36): 34253-34258 (2003)). First, TGFβ promotes PIAS4'sinteraction with SMAD3 and SMAD4 to form a ternary complex PIAS4 isknown to possess small ubiquitin-like modifier (SUMO) E3 ligase activitywithin its RING-type domain (Imoto et al., “The RING domain of PIASy isinvolved in the suppression of bone morphogenetic protein-signalingpathway,” Biochemical and biophysical research communications, 319(1):275-282 (2004)), so it promotes the sumoylation of SMAD3, in turnstimulating its nuclear export and inhibiting SMAD3/4 driventranscription (Imoto et al., “Sumoylation of SMAD3 stimulates itsnuclear export during PIASy-mediated suppression of TGF-beta signaling,”Biochem Biophys Res Commun, 370(2): 359-365 (2008); Lee et al.,“Sumoylation of SMAD4, the common Smad mediator of transforming growthfactor-beta family signaling,” The Journal of biological chemistry,278(30): 27853-27863 (2003)). Furthermore, PIAS4 directly recruits andinteracts with histone deacetylase 1 (HDAC1) to repress SMAD3 driventranscriptional activation. In all, PIAS4 is an important negativeregulator of TGFβ signaling.

Protein ubiquitination is the major protein processing function incells. Ubiquitin (Ub) flags a targeted protein for degradation throughthe 26 s proteasome or lysosome (Tanaka et al., “c-Cbl-dependentmonoubiquitination and lysosomal degradation of gp130,” Mol Cell Biol.,28(15): 4805-4818 (2008)). Ubiquitin is conjugated to a target proteinin a three-step process. First, an E1 ubiquitin-activating enzyme bindsto ubiquitin via a thioester covalent bond. Then, the E1 transfers theubiquitin to an E2 ubiquitin-conjugating enzyme. Finally, the C-terminusof Ub is attached to the ε-amino lysine (K) residue of the substrate,mediated by a ubiquitin E3 ligase. There are several families of theseubiquitin E3 ligases that include over 1,000 proteins (Jin et al., “DualE1 activation systems for ubiquitin differentially regulate E2 enzymecharging,” Nature, 447(7148): 1135-1138 (2007); Hatakeyama et al., “Ubox proteins as a new family of ubiquitin-protein ligases,” J Biol Chem,276(35): 33111-33120 (2001)). Of these, the E6-AP Carboxyl Terminus(HECT) domain E3 ligase family remains poorly characterized (Rotin etal., “Physiological functions of the HECT family of ubiquitin ligases,”Nature reviews Molecular cell biology, 10(6): 398-409 (2009)). There are˜30 HECT E3 ligases in mammalian cells, and functional data is onlyavailable for a select few including E6AP, Smurf, and NEDD4. HECT E3ligases possess a unique feature in which they accept ubiquitin from anE2 ubiquitin-conjugating enzyme in the form of a thioester bond anddirectly transfer the ubiquitin to the substrate. An active site withinthe C-terminal of the HECT domain containing a cysteine residue isrequired for ubiquitin-thiolester formation (Huibregtse et al., “Afamily of proteins structurally and functionally related to the E6-APubiquitin-protein ligase,” Proceedings of the National Academy ofSciences of the United States of America, 92(7): 2563-2567 (1995)). Arecently identified member of the HECT E3 ligases, AREL1 encoded by theKIAA0317 gene, regulates the ubiquitination of the apoptosis proteinsSMAC, HtrA2, and ARTS (Kim et al., “Identification of a novelanti-apoptotic E3 ubiquitin ligase that ubiquitinates antagonists ofinhibitor of apoptosis proteins SMAC, HtrA2, and ARTS,” J Biol Chem,288(17): 12014-12021 (2013)).

Thus, there remains a need in the art to discover the role of theprotein encoded by KIAA0317 in various signaling pathways. Futhermore,there remains a need in the art to develop new antagonists that exertpotent anti-fibrotic activity for treating idiopathic pulmonary fibrosis(IPF). The present invention satisfies these needs.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a compound represented byFormula (I):

wherein R is

and wherein: (1) W is selected from the group consisting of H,optionally-substituted alkyl, optionally-substituted alkoxy,optionally-substituted aryl, optionally-substituted cycloalkyl,optionally-substituted heterocyclic, halogen, amino, and hydroxy; (2) Xis selected from the group consisting of H, optionally-substitutedalkyl, optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, and hydroxy; (3) Y is selected from the group consistingof H, optionally-substituted alkyl, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted, heterocyclicmoieties; and (4) Z is selected from the group consisting of H,optionally-substituted alkyl, optionally-substituted aryl,optionally-substituted cycloalkyl, and optionally-substitutedheterocyclic moieties; and wherein: (a) Y and Z optionally bind togetherto form a ring; (b) R′ is selected from the group consisting of H,optionally-substituted alkyl, optionally-substituted aryl,optionally-substituted cycloalkyl, and optionally-substitutedheterocyclic; (c) R″ is independently selected from the group consistingof H, optionally-substituted alkyl, optionally-substituted aryl,optionally-substituted cycloalkyl, and optionally-substitutedheterocyclic; wherein one or more of the alkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moieties may be substituted by oneor more C₁-C₆ alkoxy, halogen or deuterium.

In another embodiment, W is selected from the group consisting of H,optionally-substituted alkyl, and optionally-substituted aryl; wherein Xis selected from the group consisting of H, optionally-substitutedalkyl, and optionally-substituted aryl; and wherein W and X cannot bothbe H.

In another embodiment, Y is selected from the group consisting of H,optionally-substituted alkyl, and optionally-substituted aryl; wherein Zis selected from the group consisting of H, optionally-substitutedalkyl, and optionally-substituted aryl; and wherein Y and Z cannot bothbe H.

In another embodiment, R′ is H. In another embodiment, R″ is H. Inanother embodiment, R is

In another embodiment, R is

In another embodiment, R is

In another embodiment, R is

Other aspects of the present invention include a pharmaceuticalcomposition comprising a compound of any one of the prior embodimentsand at least one pharmaceutically acceptable excipient.

Another aspect of the present invention includes a method of treatingidiopathic pulmonary fibrosis (IPF) in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of a compound of any of the prior embodiments, or apharmaceutically acceptable salt thereof, wherein the compound iscapable of inhibiting TGFβ pathway and the compound binds to FIEL1.

Yet another aspect of the present invention includes methods of treatinga disease associated with an activated inflammatory pathway such as theNF-kB, TGFβ and JAK/STAT pathway, comprising administering to a subjectin need thereof a therapeutically effective amount of a compound of anyof the prior embodiments, or a pharmaceutically acceptable salt thereof,wherein the compound is capable of inhibiting NF-kB, TGFβ and/orJAK/STAT pathways, and wherein the compound binds to FIEL1. In someembodiments, the disease associated with NF-kB, TGFβ and JAK/STATpathway is selected from a group consisting asthma, chronic obstructivelung disease, pulmonary fibrosis, pneumonitis, pneumonia, cysticfibrosis, psoriasis, arthritis/rheumatoid arthritis, rhinitis,pharyngitis, cystitis, prostatitis, dermatitis, allergy, nephritis,conjunctivitis, encephalitis, meningitis, opthaltnitis, uveitis,pleuritis, pericarditis, myocarditis, atherosclerosis, multiplesclerosis, human immunodeficiency virus related inflammation, diabetes,osteoarthritis, psoriatic arthritis, inflammatory bowel disease,colitis, sepsis, vasculitis, bursitis, connective tissue disease,autoimmune disease, viral or influenza-induced inflammation, or edema.In some embodiments, the therapeutic effective amount of a compound ofany prior embodiment is about 0.1-about 20 mg/kg/d. In some embodiments,the administration of the compound of any of the prior embodiment is viaany pharmaceutically acceptable method, including but not limited tooral, inhalation, intravenous, or intramuscular.

The foregoing general description and following description of thedrawings and detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.Other objects, advantages, and novel features will be readily apparentto those skilled in the art from the following brief description of thedrawings and detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-H show in vitro and in vivo studies of FIEL1 regulating PIAS4in pulmonary fibrosis pathway. FIG. 1A shows Immunoblots of levels ofPIAS4 proteins and V5 after KIAA0317 (823aa, AREL1) and (789aa, FIEL1)plasmid expression (n=2). FIG. 1B shows immunoprecipitation of PIAS4protein from cell lysate using a PIAS4 antibody and coupled to proteinA/G beads. PIAS4 beads were then incubated with in vitro synthesizedproducts expressing HIS-V5-FIEL1 (789aa) or HIS-V5-AREL1 (823aa). Afterwashing, proteins were eluted and processed for V5 immunoblotting (n=2).FIG. 1C shows an in vitro ubiquitination assay. Purified E1 and E2components were incubated with V5-PIAS4, FIEL1, and the full complementof ubiquitination reaction components (second lane) showedpolyubiquitinated PIAS4 proteins (n=3). FIGS. 1D-E. PIAS4 protein inmurine lung epithelial cells (MLE) half-life determination with emptyplasmid (E) or FIEL1 expression (top panel); PIAS4 protein half-lifedetermination with CON shRNA or KIAA0317 shRNA expression (bottom panel)(n=3). FIG. 1F shows the results of treatment of MRC5 cells with TGFβ ina time or dose dependent manner; cells were collected and immunoblotedfor FIEL1, PIAS4, and Actin. Endogenous FIEL1 was alsoimmunoprecipitated and immunobloted for PIAS4 (n=3). FIG. 1G shows theresults of immunoblotting of PIAS4 and FIEL1 from five control and fiveIPF patient lungs. PIAS4 and both the shorter and longer forms ofKIAA0317 were quantified using ImageJ and graphed. FIG. 1H shows theresults of treatment of C57BL/6J mice with bleomycin (0.02 U) for up to21 days. Mice were then euthanized, and lungs were isolated and assayedfor PIAS4 and FIEL1 immunoblotting. Bands corresponding to each proteinon immunoblots were quantified using ImageJ software, and the resultsare displayed graphically. n=4, *p<0.05 compared to day 0 (t-test).

FIGS. 2A-J show SMAD reporter assays suggesting that FIELS promotes TGFβsignaling. FIGS. 2A& F SMAD reporter assays. 293T cells wereco-transfected with Cignal SMAD dual luciferase reporter plasmids alongwith Empty (E), FIEL1, con shRNA, or KIAA0317 shRNA. 24 h later, cellswere treated with TGFβ for 2-18 h. Cells were collected and assayed forluciferase activity to evaluate SMAD promoter activity (n=3). FIGS. 2B&G293T cells were transfected with Empty (E), FIEL1, CON shRNA, orKIAA0317 shRNA for 48 h before TGFβ treatment (0-2 ng/ml) for 1 h. Cellswere then collected and immunobloted. Cell lysates wereimmunoprecipitated using SUMO antibody before SMAD2, 3, and 4immunoblotting (n=2). FIGS. 2C&H. 293T cells were transfected with Empty(E), FIEL1, CON shRNA, or KIAA0317 shRNA for 48 h before TGFβ treatment(2 ng/ml) for up to 1 h. Cells were then collected and nuclear/cytosolfractions were isolated before being immunobloted. FIGS. 2D&I. MRC5cells were transfected with Empty (E), FIEL1, CON shRNA, or KIAA 0317shRNA for 48 h before TGFβ dose course treatment for an additional 18 h.Cells were then collected and immunobloted (n=2). FIGS. 2E&J. MRC5 cellswere seeded in 35 mm glass bottom dishes before being transfected withEmpty (E), FIEL1, CON shRNA, or KIAA0317 shRNA for 48 h before TGFβtreatment for an additional 18 h. Cells were then fixed andimmunostained with α-SMA and FN antibodies. The nucleus wascounterstained with DAPI (n=3).

FIGS. 3A-J shows that PIAS4 phosphorylation by PKCξ is required forFIEL1 binding. FIG. 3A. Endogenous PIAS4 was immunoprecipitated andimmunoblotted for Erk1, PKCα, and PKCξ (n=2). FIG. 3B. MLE cells weretransfected with increasing amounts of PKCξ or JNK1 plasmids for 18 hbefore PIAS4 immunoblotting (n=2). FIG. 3C. PIAS4 protein half-lifedetermination with CON shRNA or PKCξ shRNA expression (n=3). FIG. 3D.PIAS4 protein half-life determination with Empty or PKCξ plasmidoverexpression (n=3). FIGS. 3E-F. MRC5 cells were treated with TGFβ in atime or dose dependent manner; cells were collected and immunoblottedfor FIEL1, PIAS4, PKCξ, p-PKCξ, and Actin. Endogenous PIAS4 was alsoimmunoprecipitated and followed by PKCξ, PKCα, phospho-serine, andphospho-threonine immunoblotting (n=2). FIG. 3G. In vitro PKCξ kinaseassay using PIAS4 as the substrate. *heat inactivated PKCξ (n=2). FIG.3H. Immunoblots showing levels of FIEL1, PKCξ, p-PKCξ, and PIAS4 proteinin 293T cells transfected with either con shRNA or PKCξ shRNA followedby a TGFβ dose treatment. Endogenous PIAS4 was also immunoprecipitatedand followed by PKCξ, phospho-serine, and phospho-threonineimmunoblotting. FIG. 3I. 293T cells were transfected with WT, S14A,S18A, or S14/18A PIAS4 before being treated with a dose course of TGFβ.Cells were then collected and assayed for V5-PIAS4. OverexpressedV5-PIAS4 was also immunoprecipitated using a V5 antibody and followed byphospho-serine immunoblotting (n=2). FIG. 3J. Four biotin labeled PIAS4peptides were bound to streptavidin and served as the bait for FIEL1binding. After washing, proteins were eluted and immunobloted forFIEL1-V5 (n=2).

FIGS. 4A-K shows that GSK3β phosphorylation of FIEL1 is required forPIAS4 targeting, and FIEL1 residues P779 and phosphorylated T783 areboth required for PIAS4 interaction. FIG. 4A. Endogenous PIAS4 wasimmunoprecipitated and followed by JNK2, PKCα, and GSK3β antibodyimmunoblotting (n=2). FIG. 4B. MLE cells were transfected withincreasing amounts of WT or constitutively activated GSK3β hyper mutantplasmids for 18 h before PIAS4 immunoblotting. The arrow indicates theoverexpressed GSK3β (n=2). FIG. 4C. PIAS4 protein half-lifedetermination with WT GSK3β or Hyperactive GSK3β plasmid overexpression.The arrow indicates the overexpressed GSK3β (n=2). FIG. 4D. MRC5 cellswere treated with TGFβ in a time or dose dependent manner; cells werethen collected and immunobloted for FIEL1, PIAS4, and Actin. EndogenousFIEL1 was also immunoprecipitated and followed by phospho-serine andphospho-threonine immunoblotting (n=2). FIG. 4E. Immunoblots showinglevels of GSK3β, PIAS4, and FIEL1 protein in 293T cells transfected witheither CON shRNA or GSK3β shRNA followed by a TGFβ dose treatment.Endogenous PIAS4 was also immunoprecipitated and followed byphospho-serine and phospho-threonine immunoblotting. FIG. 4F. Lungsamples from FIG. 1F were subjected to FIEL1 immunoprecipitation,followed by PIAS4 and phospho-threonine immunoblotting. FIG. 4G. Invitro GSK3β kinase assay using PIAS4 as the substrate. *heat inactivatedGSK3β (n=2). FIG. 4H. 293T cells were transfected with Empty, WT, orT783A FIEL1 for 24 h. Cells were then collected and immunobloted forV5-FIEL1 and PIAS4. Overexpressed V5-FIEL1 was also immunoprecipitatedusing V5 antibody and followed by phospho-threonine immunoblotting(n=2). FIG. 4I. Four biotin labeled FIEL1 peptides were pre-bound tostreptavidin and served as the bait for PIAS4 binding. After washing,proteins were eluted and processed for PIAS4 immunoblotting (n=2). FIG.4J. PIAS4 Peptide 2 (Biotin-MSFRVS(p)DLQM) was pre-bound to streptavidinand served as the bait for FIEL1 binding. After washing, proteins wereeluted and processed for FIEL1 immunoblotting (n=2). FIG. 4K. FIEL1Peptide 2 (Biotin-QIIAAPTHST(p)LPTA) was bound to streptavidin andserved as the bait for PIAS4 binding. After washing, proteins wereeluted and processed for PIAS4 immunoblotting (n=2).

FIGS. 5A-I shows that in vivo expression of FIEL1 exacerbates bleomycininduced lung injury. C57BL/6J mice were administered i.t. withLenti-Empty or Lenti-FIEL1 (10⁷ PFU/mouse) for 144 h; mice were thenadministered i.t. with bleomycin (0.02 U) for 1-21 days. Mice wereeuthanized, and lungs were lavaged with saline, harvested, and thenhomogenized. Lavage protein and CXCL1 concentrations were measured inFIGS. 5A-B. Lavage total cell counts were measured in (FIG. 5C). Lavagecells were then processed for Wright-Giemsa stain; Lavage macrophages,neutrophils, and lymphocytes were counted and graphed (FIGS. 5D-F). FIG.5G. Survival studies of mice that were given bleomycin. Mice werecarefully monitored over time, moribund, preterminal animals wereimmediately euthanized and recorded as deceased. Kaplan-Meier survivalcurves were generated using SPSS software (p<0.05). Empty: n=9, FIEL1:n=11. FIG. 5H. Hydroxyproline contents were measured in lungs from 7,14, and 21 d after bleomycin challenge. I. H&E and Trichrome stainingwas performed on lung samples. Original magnification, ×10. *p<0.05compared to Empty (t-test). Data are an average of two experiments (A-I,4-6 mice/group).

FIGS. 6A-I shows that a decrease in expression of FIEL amelioratesbleomycin-induced lung injury in vivo. C57BL/6J mice were administeredi.t. with Lenti-CON shRNA or Lenti-KIAA0317 shRNA (10⁷ PFU/mouse) for144 h; mice were then administered i.t. with bleomycin (0.05 U) for 1-21days. Mice were euthanized, and lungs were lavaged with saline,harvested, and then homogenized. Lavage protein and CXCL1 concentrationswere measured in FIGS. 6A-B. Lavage total cell counts were measured in(C). Lavage cells were then processed for Wright-Giemsa stain; Lavagemacrophages, neutrophils, and lymphocytes were counted and graphed(FIGS. 6D-F). FIG. 6G. Survival studies of mice that were givenbleomycin. Mice were carefully monitored over time; moribund,preterminal animals were immediately euthanized and recorded asdeceased. Kaplan-Meier survival curves were generated using SPSSsoftware (p<0.05). CON shRNA: n=8, KIAA0317 shRNA: n=8. FIG. 6H.Hydroxyproline contents were measured in lungs from 7, 14, and 21 dafter bleomycin challenge. I. FIGS. 6H&E and Trichrome staining wasperformed on lung samples. Original magnification, ×10. *p<0.05 comparedto Empty (t-test). Data are an average of two experiments (A-I,4-6-mice/group).

FIGS. 7A-I shows a FIEL inhibitor binding site, and inhibition of FIEL1and PIAS4 interaction by representative inhibitors (BE 1480 and BC1485). FIG. 7A. Structural analysis of the FIEL1 HECT domain revealed amajor cavity within the c-terminal of the HECT domain. FIG. 7B.Structures of the BC-1480 backbone and lead compound BC-1485. FIG. 7C.FIEL1 protein was HIS-purified from FIEL1 expression in 293T cells usingcobalt beads. Beads were then extensively washed prior to exposure toBC-1480 or BC-1485 at different concentrations (10⁻⁴ to 100 μM).Purified PIAS4 protein was then incubated with drug-bound FIEL1 beadsovernight. Beads were washed, and proteins were eluted and resolved onSDS-PAGE. The relative amounts of PIAS4 detected in the pull-downs wasnormalized to loading and quantified (n=2). FIG. 7D. MLE cells wereexposed to BC-1480 or BC-1485 at various concentrations for 18 h. Cellswere then collected and immunoblotted. FIGS. 7E-J. C57BL/6J mice wereadministered with bleomycin (0.05 U) for 7-21 days. Compounds BC-1480and BC-1485 were given to mice through drinking water with an estimateddose of 5 mg/kg/d. Mice were euthanized, and lungs were lavaged withsaline, harvested, and then homogenized. Lavage proteins, CXCL1, andtotal cell count were measured in FIGS. 7E-G. H. Survival studies ofmice that were given bleomycin and compound treatments. Mice werecarefully monitored over time; moribund, preterminal animals wereimmediately euthanized and recorded as deceased. Kaplan-Meier survivalcurves were generated using SPSS software (p<0.01 compared to Vehicle).Vehicle: n=24, BC-1480: n=13, BC-1485: n=12. FIG. 7I. Hydroxyprolinecontents were measured in lungs from 7, 14, and 21 d after bleomycinchallenge. FIG. 7J. Trichrome staining was performed on lung samplesfrom E. Original magnification, ×10. *p<0.05 compared to Vehicle(t-test). Data are an average of two experiments (FIGS. 7E-J, 4-8mice/group)

FIG. 8 shows molecular regulation of TGFβ signaling mediated by FIEL1.

FIGS. 9A-G shows that the expression of FIEL1 reduces the half-life ofPIAS4. FIG. 9A. PIAS4 protein half-life study in MLE cells transfectedwith Empty plasmid or Ubiquitin plasmid. FIG. 9B. PIAS4 proteinhalf-life study with MG132 or Leupeptin. FIG. 9C. Immunoblots showinglevels of PIAS4 proteins and V5 after ectopic FIEL1 or UBE3B plasmidexpression. FIG. 9D. Immunoblots showing levels of FIEL1 proteins afterectopic FIEL1 plasmid expression in MLE, HeLa, and 293T cells. FIG. 9E.Immunoblots showing levels of PIAS4 proteins and V5 after ectopic FIEL1plasmid expression in HeLa and 293T cells. FIG. 9F. MLE cells weretransfected with an inducible control or FIEL1 plasmid under control ofexogenous doxycycline. Cells were treated with doxycycline in a dosedependent manner. Cells were then collected and cell lysates wereimmunoblotted for FIEL1 and PIAS4. FIG. 9G. MRC5 cells were exposed toTGFβ for 24 h before being assayed for PIAS4 and FIEL1 proteinhalf-lives.

FIGS. 10A-C shows that the expression of FIEL1 does not reduce thehalf-life of PIAS4 with K31R mutation. FIG. 10A. Several point mutantsof PIAS4 were designed and cloned into a pcDNA3.1D/V5-HIS vector. FIG.10B. Half-life study of WT, K31R, K35R, and K114R PIAS4 in MLE cells. C.MLE cells were co-transfected with WT or PIAS4 lysine mutants with orwithout HA-Ubiquitin. Cells were then collected and immunobloted. PIAS4protein levels were quantified and graphed n=3.

FIGS. 11A-E shows an alanine scan study of PIAS4, which suggests thatboth S18 and Q21 residues are important for FIEL1 interaction. FIG. 11A.Several deletional mutants of PIAS4 were designed and cloned into apcDNA3.1D/V5-HIS vector. FIGS. 11B-C. FIEL1 protein wasimmunoprecipitated from cell lysates using a KIAA0317 antibody andcoupled to protein A/G beads. FIEL1 beads were then incubated with invitro synthesized products expressing HIS-V5-PIAS4 mutants. Afterwashing, proteins were eluted and processed for V5-PIAS4 immunoblotting.FIG. 11D. Half-life study of WT, Q21A, S14A, S18A, S18A/Q21A, andS14A/S18A PIAS4 in MLE cells. FIG. 11E. MLE cells were co-transfectedwith WT or PIAS4 mutants with or without FIEL1. Cells were thencollected and immunobloted.

FIGS. 12A-H shows studies of PIAS4 mutations, which suggests that bothQ21 residue and PKCξ phospharylated S18 residue are required for FIEL1binding. FIG. 12A. Several deletional mutants of FIEL1 were designed andcloned into a pcDNA3.1D/V5-HIS vector. FIGS. 12B-F, H. PIAS4 protein wasimmunoprecipitated from cell lysate using a PIAS4 antibody and coupledto protein A/G beads. PIAS4 beads were then incubated with in vitrosynthesized products expressing HIS-V5-FIEL1 mutants. After washing,proteins were eluted and processed for V5-FIEL1 immunoblotting. FIG.12G. MLE cells were transfected with WT or FIEL1 mutants. Cells werethen collected and immunoblotted for PIAS4 protein. FIG. 12I. Thecartoon illustrates the “double locking” molecular interplay betweenPIAS4 and FIEL1. Specifically, both the P779 and GSK3β phosphorylatedT783 residues within FIEL1 are required for PIAS4 binding; both the Q21and PKCξ phosphorylated S18 residues within PIAS4 are required for FIEL1binding.

FIGS. 13A-H shows studies of FIEL1 mutations, which suggests that FIEL1residues P779 and GSK3β phosphorylated T783 are both required for PIAS4interaction. FIG. 13A. PIAS4 protein half-life determination with CONshRNA or GSK3β shRNA expression. FIG. 13B. MLE cells were transfectedwith increasing amounts of WT, T783A, or T783D mutant FIEL1 plasmids for18 h before PIAS4 immunoblotting. FIG. 13C. PIAS4 protein half-lifedetermination with WT, T783A, or T783D mutant FIEL1. FIG. 13D. MLE cellswere transfected with increasing amounts of WT, T783A, P779L, orT783A/P779L double mutant FIEL1 plasmids for 18 h before PIAS4immunoblotting. FIG. 13E. PIAS4 protein half-life determination with WT,T783A, P779L, or T783A/P779L double mutant FIEL1. FIG. 13F. 293T cellswere transfected with WT, T783A, P779L, or T783A/P779L double mutantFIEL1 before being treated with TGFβ. Cells were then collected andassayed for PIAS4. FIG. 13G. MLE cells were transfected with increasingamounts of WT, I514V or D207V mutant FIEL1 plasmids for 18 h beforePIAS4 immunoblotting. FIG. 13H. PIAS4 protein half-life determinationwith WT, I514V, or D207V mutant FIEL1 expression.

FIG. 14 shows representative inhibitors of FIEL1.

FIGS. 15A-G shows the synthesis of FIEL1 inhibitor BC1485, and itsinhibition of TGFβ pathway. FIG. 15A. Diagram of the BC-1485 synthesisprocess. FIGS. 15B-C. Docking studies of the lead compound, BC-1485,interacting with the FIEL1-HECT domain. FIG. 15D. MRC5 cells weretreated with the compound at various doses; cells were also co-treatedwith TGFβ at 2 ng/ml. 18 h later, cells were collected and assayed forα-SMA expression. FIG. 15E. MRC5 cells were seeded in 35 mm glass bottomdishes before co-treatment with TGFβ at 2 ng/ml and BC-1480 or BC1485 at5 μM. FIG. 15F. PIAS4 protein half-life determination after BC-1480 orBC1485 treatment at 5 μM for 18 h. FIG. 15G, PIAS4 and KIAA0317 mRNAanalysis after BC1485 treatment for 18 h.

FIGS. 16A-E shows BC1480 and BC 1485 inhibition of pulmonary fibrosis ina mouse model. C57BL/6J mice were administered i.t. with bleomycin (0.05U) for 7-21 days. Compounds BC-1480 and BC-1485 were given to micethrough drinking water with an estimated dose of 5 mg/kg/d. Mice werethen euthanized, and lungs were lavaged with saline, harvested, and thenhomogenized. Lavage IL-6 was measured in (FIG. 16A). Lavage cells werealso processed for Wright-Giemsa stain; Lavage macrophages, neutrophilsand lymphocytes were counted and graphed (FIGS. 16B-D). H&E staining wasperformed on lung samples from A. Original magnification, ×10. *p<0.05compared to Vehicle (t test). Data are an average of two experiments(A-E, 4-8 mice/group).

FIGS. 17A-E shows anti-inflammatory activity of a FIEL1 small moleculeinhibitor in Pseudomonas pneumonia model. C57BL/6J mice wereadministered i.t. with PA103 (104 PFU/mouse). BC-1365 was given throughIP injection (10 mg/kg) at the same time. 18 h later, mice weresacrificed, and lungs were lavaged with saline, harvested, and thenhomogenized. Lavage protein, cell count, bacterial count, and cytokinesecretion were measured in (FIGS. 17A-D). FIG. 17E. H&E staining wasperformed on lung samples from FIG. 17A (Original magnification, ×10).Data are an average of two experiments (A-E, n=5-10 mice/group).

FIGS. 18A-E shows anti-inflammatory activity of a FIEL1 small moleculeinhibitor in LPS pneumonia model. C57BL/6J mice were administered i.t.with LPS (E. coli, 3 mg/kg). BC-1365 was given through IP injection (10mg/kg) at the same time. 18 h later, mice were sacrificed, and lungswere lavaged with saline, harvested, and then homogenized. Lavageprotein, cell count and cytokine secretion were measured in (FIGS.18A-D). FIG. 18E. H&E staining was performed on lung samples from FIG.18A (Original magnification, ×20) A-E, n=4 mice/group.

FIG. 19 shows that BC1485 inhibits JAK/STAT pathway through decreasingJAK1 and JAK2, p-Stat1 and p-Stat3 protein levels.

FIG. 20 shows that BC1485 reduces dextran sulfate sodium (DSS) inducedacute colonic inflammation. C57BL6 mice were fed with water ad libcontaining 2.5% dextran sulfate sodium (DSS) for up to 7 days. Mice weretreated with either vehicle (control) or BC-1261 (administered intodrinking water at 30 ug/ml, ˜5 mg/kg/d dosing). Mice were monitoreddaily weights were measured and graphed, shown in FIG. 20A. Mice werethen euthanized, the length of the colon and weight of the spleen weremeasured and graphed, shown in FIGS. 20B-D. Colonic tissues were alsoanalysed for TNFα, IL1 and IL6 shown in FIGS. 20E H&E staining wasperformed on colonic sections. The data in FIG. 20F represent n=6mice/group, *P<0.05 versus DSS and **P<0.05 versus control. The resultsshows that BC1485 effectively reduces dextran sulfate sodium inducedacute colonic inflammation in mice. FIG. 20G provides pictures of thetissues at interest.

DETAIL DESCRIPTION

I. Compounds

Compounds of the present disclosure include novel compounds representedby Formula (I):

Wherein R is:

and wherein:

-   (1) W is selected from the group consisting of H,    optionally-substituted alkyl, optionally-substituted alkoxy,    optionally-substituted aryl, optionally-substituted cycloalkyl,    optionally-substituted heterocyclic, halogen, amino, and hydroxy;-   (2) X is selected from the group consisting of H,    optionally-substituted alkyl, optionally-substituted alkoxy,    optionally-substituted aryl, optionally-substituted cycloalkyl,    optionally-substituted heterocyclic, halogen, amino, and hydroxy;-   (3) Y is selected from the group consisting of H,    optionally-substituted alkyl, optionally-substituted aryl,    optionally-substituted cycloalkyl, optionally-substituted    heterocyclic moieties;-   (4) Z is selected from the group consisting of H,    optionally-substituted alkyl, optionally-substituted aryl,    optionally-substituted cycloalkyl, and optionally-substituted    heterocyclic moieties;    and wherein Y and Z optionally bind together to form a ring;-   (5) R′ is selected from the group consisting of H,    optionally-substituted alkyl, optionally-substituted aryl,    optionally-substituted cycloalkyl, and optionally-substituted    heterocyclic;-   (6) R″ is independently selected from the group consisting of H,    optionally-substituted alkyl, optionally-substituted aryl,    optionally-substituted cycloalkyl, and optionally-substituted    heterocyclic;    wherein one or more of the alkyl, cycloalkyl, heterocycloalkyl, aryl    or heteroaryl moieties may be substituted by one or more C₁-C₆    alkoxy, halogen or deuterium.

In another embodiment, W is selected from the group consisting of H,optionally-substituted alkyl, and optionally-substituted aryl; wherein Xis selected from the group consisting of H, optionally-substitutedalkyl, and optionally-substituted aryl; and wherein W and X cannot bothbe H. In some embodiments, W is selected from the group consisting of Hand C₁-C₆ alkyl.

In another embodiment, Y is selected from the group consisting of H,optionally-substituted alkyl, and optionally-substituted aryl; wherein Zis selected from the group consisting of H, optionally-substitutedalkyl, and optionally-substituted aryl; and wherein Y and Z cannot bothbe H. In some embodiments, X is selected from the group consisting of H,C₁-C₆ alkyl, and (C₀-C₆)alkyl(C₆-C₁₂)aryl.

In another embodiment, R′ is H or R′ is selected from the groupconsisting of H and C₁-C₆ alkyl, and R″ is selected from the groupconsisting of H and C₁-C₆ alkyl. In another embodiment, R″ is H. Inanother embodiment, R is

In another embodiment, R is

In another embodiment, R is

In another embodiment, R is

Specific embodiments of compounds of the present invention include thecompounds in FIG. 14. Additional embodiments are indicated in Table 1,below.

TABLE 1

R′ R″ W X Y Z H H H C₁—C₆ alkyl H

C₁—C₆ alkyl H H C₁—C₆ alkyl H

H H H C₁—C₆ alkyl C₁—C₆ alkyl C₁—C₆ alkyl C₁—C₆ alkyl H H C₁—C₆ alkylC₁—C₆ alkyl C₁—C₆ alkyl H H H C₁—C₆ alkyl

C₁—C₆ alkyl H H C₁—C₆ alkyl

H H H C₁—C₆ alkyl

H H H

H C₁—C₆ alkyl H H H

C₁—C₆ alkyl C₁—C₆ alkyl H H H C₁—C₆ alkyl H adamantyl H H H C₁—C₆ alkylH

H H H C₁—C₆ alkyl H

Other embodiments include one or more of the embodiments in Table 1,wherein one of the defined moieties is substituted by alkoxy, halogen,amino, or hydroxy. In another embodiment, when the X, Y, Z moietycontains a phenyl moiety it may be substituted by one or more of alkoxy,halogen, amino, or hydroxy. In another embodiment, one or more of themoieties in Table 1 is substituted by a C₁-C₆ alkoxy, halogen ordeuterium.

II. Methods of Treatment

One aspect of the present technology includes methods of treatingidiopathic pulmonary fibrosis (IPF) in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of a compound, or a pharmaceutically acceptable salt thereof,wherein the compound is capable of inhibiting TGFβ pathway, and whereinthe compound binds to FIEL1.

In some embodiments, the compound, or a pharmaceutically acceptable saltthereof, capable of inhibiting TGFβ pathway, and binding to FIEL1 isrepresented by Formula (I) or is represented by any other embodimentdisclosed in the section titled “Compounds of the invention.”

The subject being treated may be a mammal, for example, in a preferredembodiment the subject is a human.

Another aspect of the present technology includes methods of treating adisease associated with an activated inflammatory pathway such as NF-kB,TGFβ and JAK/STAT pathway comprising administering to a subject in needthereof a therapeutically effective amount of a compound, or apharmaceutically acceptable salt thereof, wherein the compound iscapable of inhibiting NF-kB, TGFβ and/or JAK/STAT pathways, and whereinthe compound binds to FIEL1, and wherein the compound is represented byFormula (I). In one embodiment the subject is a human.

Another aspect of the present technology includes methods of treating adisease associated with an activated inflammatory pathway such as NF-kB,TGFβ and JAK/STAT pathway, wherein the disease associated with theactivated inflammatory pathway such as NF-kB, TGFβ and JAK/STAT pathwayis selected from the group consisting of asthma, chronic obstructivelung disease, pulmonary fibrosis, pneumonitis, pneumonia, cysticfibrosis, psoriasis, arthritis/rheumatoid arthritis, rhinitis,pharyngitis, cystitis, prostatitis, dermatitis, allergy, nephritis,conjunctivitis, encephalitis, meningitis, opthahnitis, uveitis,pleuritis, pericarditis, myocarditis, atherosclerosis, multiplesclerosis, human immunodeficiency virus related inflammation, diabetes,osteoarthritis, psoriatic arthritis, inflammatory bowel disease,colitis, sepsis, vasculitis, bursitis, connective tissue disease,autoimmune disease, viral or influenza-induced inflammation, or edema.

Compounds represented by Formula 1, or pharmaceutically acceptable saltsor solvates thereof, or a composition comprising such a compound or apharmaceutically acceptable salt or solvate thereof, can be administeredto a patient or subject in need of treatment either individually, or incombination with other therapeutic agents that have similar orsynergistic biological activities. Additionally, Formula I compounds andcompositions can be administered as a single dose or as multiple dailydoses by a practicing medical practitioner.

A composition comprising a compound of the present disclosure may beadministered to individuals in a formulation with one or morepharmaceutically acceptable excipient(s). Wide varieties ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy”, 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds7th ed., Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. PharmaceuticalAssoc.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredtherapeutic effect. Thus, the agent can be incorporated into a varietyof formulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols. As such, administration of theagents can be achieved in various ways, including but not limited tooral, buccal, rectal, parenteral, intraperitoneal, intradermal, topical,pulmonary, nasal, inhalation, transdermal, intracheal, etc.,administration.

The dosage administered will be dependent upon multiple factors, such asthe age, health, weight, and/or disease state of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and/or the natureand magnitude of the biological effect desired. An exemplary dosage maybe about 0.1-about 20 mg/kg/d, or any amount inbetween these twoamounts. Other exemplary dosages include, but are not limited to, about0.1 about 10 mg/kg/d or about 0.5-about 10 mg/kg/d. Still otherexemplary dosages include, but are not limited to, about 0.1, about 0.2,about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14,about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about17.5, about 18, about 18.5, about 19, about 19.5, or about 20 mg/kg/d.When combination therapy is used, the compound and the other therapeuticagent can be administered separately at different time intervals, orsimultaneously.

III. Pharmaceutical Formulations

Pharmaceutical compositions and medicaments may be prepared by mixingone or more compounds of the invention, prodrugs thereof,pharmaceutically acceptable salts or solvates thereof, stereoisomersthereof, tautomers thereof, or solvates thereof, with pharmaceuticallyacceptable carriers, excipients, binders, diluents or the like to treatidiopathic pulmonary fibrosis (IPF) or a disease associated withactivated inflammatory pathway such as NF-kB, TGFβ and JAK/STAT pathway.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredtherapeutic effect Thus, the agent can be incorporated into a variety offormulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

The compounds and compositions of the invention may be used to prepareformulations and medicaments that prevent or treat idiopathic pulmonaryfibrosis (IPF), as described herein. Other diseases or disorders can betreated include inflammatory disorders such as asthma, chronicobstructive lung disease, pulmonary fibrosis, pneumonitis, pneumonia,cystic fibrosis, psoriasis, arthritis/rheumatoid arthritis, rhinitis,pharyngitis, cystitis, prostatitis, dermatitis, allergy, nephritis,conjunctivitis, encephalitis, meningitis, opthalmitis, uveitis,pleuritis, pericarditis, myocarditis, atherosclerosis, multiplesclerosis, human immunodeficiency virus related inflammation, diabetes,osteoarthritis, psoriatic arthritis, inflammatory bowel disease,colitis, sepsis, vasculitis, bursitis, connective tissue disease,autoimmune disease, viral or influenza-induced inflammation, or edema.

Such compositions can be in any pharmaceutically acceptable form, suchas but not limited to in the form of, for example, granules, powders,tablets, capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions. The compositions can be formulated for anypharmaceutically acceptable route of administration, such as forexample, by oral, parenteral, pulmonary, topical, rectal, nasal, vaginaladministration, or via implanted reservoir. Parenteral or systemicadministration includes, but is not limited to, subcutaneous,intravenous, intraperitoneally, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjections. The following dosage forms are given by way of example andshould not be construed as limiting the invention.

Pharmaceutically acceptable salts of the invention compounds areconsidered within the scope of the present invention. The compounds ofthe invention have a number of basic nitrogen groups, and as such,pharmaceutically acceptable salts can be formed with inorganic acids(such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid,and phosphoric acid), organic acids (e.g. formic acid, acetic acid,trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, lacticacid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid)or acidic amino acids (such as aspartic acid and glutamic acid). Thecompounds of the present invention may have acidic substituent groups,and in such cases, it can form salts with metals, such as alkali andearth alkali metals (e.g. Na⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺), organicamines (e.g. ammonia, trimethylamine, triethylamine, pyridine, picoline,ethanolamine, diethanolamine, triethanolamine) or basic amino acids(e.g. arginine, lysine and ornithine).

Certain compounds within the scope of the invention are derivativesreferred to as prodrugs. The expression “prodrug” denotes a derivativeof a known direct acting drug, e.g. esters and amides, which derivativehas enhanced delivery characteristics and therapeutic value as comparedto the drug, and is transformed into the active drug by an enzymatic orchemical process; see Notari, R. E., “Theory and Practice of ProdrugKinetics,” Methods in Enzymology, 112: 309-23 (1985); Bodor, N., “NovelApproaches in Prodrug Design,” Drugs of the Future, 6: 165-82 (1981);and Bundgaard, H., “Design of Prodrugs: Bioreversible-Derivatives forVarious Functional Groups and Chemical Entities,” in DESIGN OF PRODRUGS(H. Bundgaard, ed.), Elsevier (1985), and Goodman and Gilmans, ThePharmacological Basis Of Therapeutics, 8th ed., McGraw-Hill (1992).

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds of the present invention, or pharmaceuticallyacceptable salts or tautomers thereof, with at least one additive suchas a starch or other additive. Suitable additives include anypharmaceutically acceptable excipient, including but not limited tosucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch,agar, alginates, chitins, chitosans, pectins, tragacanth gum, gumarabic, gelatins, collagens, casein, albumin, synthetic orsemi-synthetic polymers or glycerides. Optionally, oral dosage forms cancomprise other ingredients to aid in administration, such as an inactivediluent, or lubricants such as magnesium stearate, or preservatives suchas paraben or sorbic acid, or anti-oxidants such as ascorbic acid,tocopherol or cysteine, a disintegrating agent, binders, thickeners,buffers, sweeteners, flavoring agents or perfuming agents. Tablets andpills may be further treated with suitable coating materials known inthe art.

IV. Definitions

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. The claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely”, “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending uponthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. When the term “pharmaceutically acceptable” isused to refer to a pharmaceutical carrier or excipient, it is impliedthat the carrier or excipient has met the required standards oftoxicological and manufacturing testing or that it is included on theInactive Ingredient Guide prepared by the U.S. and Drug administration.

By “patient” is meant any animal for which treatment is desirable.Patients may be mammals, and typically, as used herein, a patient is ahuman individual.

The term “pharmaceutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds of the present inventionwhich are water or oil-soluble or dispersible; which are suitable fortreatment of diseases without undue toxicity, irritation, andallergic-response; which are commensurate with a reasonable benefit/riskratio; and which are effective for their intended use. The salts can beprepared during the final isolation and purification of the compounds orseparately by reacting the appropriate compound in the form of the freebase with a suitable acid. Representative acid addition salts includeacetate, adipate, alginate, L-ascorbate, aspartate, benzoate,benzenesulfonate (besylate), bisulfate, butyrate, camphorate,camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate,glutarate, glycerophosphate, glycolate, hemisuifate, heptanoate,hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isethionate), lactate, maleate, malonate,DL-mandelate, mesitylenesulfonate, methanesulfonate,naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,pamoate, pectinate, persulfate, 3-phenylproprionate, phosphonate,picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate,tartrate, L-tartrate, trichloroacetate, trifluoroacetate, phosphate,glutamate, bicarbonate, para-toluenesulfonate (p-tosylate), andundecanoate. Also, basic groups in the compounds of the presentinvention can be quaternized with methyl, ethyl, propyl, and butylchlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamylsulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, andiodides; and benzyl and phenethyl bromides. Examples of acids which canbe employed to form pharmaceutically acceptable addition salts includeinorganic acids such as hydrochloric, hydrobromic, sulfuric, andphosphoric, and organic acids such as oxalic, maleic, succinic, andcitric. Salts can also be formed by coordination of the compounds withan alkali metal or alkaline earth ion. Hence, the present inventioncontemplates sodium, potassium, magnesium, and calcium salts of thecompounds of the compounds of the present invention and the like.

An “effective” amount of an agent is meant to mean an amount of atherapeutic agent, or a rate of delivery of a therapeutic agent,effective to facilitate a desired therapeutic effect. The precisedesired therapeutic effect will vary according to the condition to betreated, the formulation to be administered, and a variety of otherfactors that are appreciated by those of ordinary skill in the art.

The term “solvates” is used in its broadest sense. For example, the termsolvates includes hydrates formed when a compound of the presentinvention contains one or more bound water molecules.

As used herein, the term “alkyl” is used in its broadest sense. Alkylgroups may be optionally substituted as defined herein. Examples ofalkyl radicals include methyl, ethyl, n-propyl, isopropyl, cyclopropyl,cyclopmpylmethyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl,pentyl, neopentyl, iso-amyl, hexyl, cyclohexyl,trans-1,2-di-ethylcyclohexyl, octyl, nonyl and the like. For example,the abbreviation “(C₁-C₆)-alkyl groups” includes (C3-C6)-cycloalkylgroups as well as straight and branched alkyl groups, and“O(C₁-C₈)-alkyl groups” includes the straight-chain O(C1-C8)-alkylgroups, branched O(C1-C6″)-alkyl groups, and cyclic O(C1-C6)-alkylgroups.

The term “alkoxy,” as used herein, refers to an alkyl ether radical,wherein the term alkyl is as defined herein. Examples of suitable alkylether radicals include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,iso-butoxy, sec-butoxy, tert-butoxy, cyclopentoxy, and the like.

The term “aryl,” as used herein, means a carbocyclic aromatic systemcontaining one, two or three rings wherein such rings may be attachedtogether in a pendent manner or may be fused. The term “aryl” embracesaromatic radicals such as phenyl, naphthyl, anthracenyl, phenanthryl,and biphenyl. The aryl groups of the present invention can be optionallysubstituted with one, two, three, four, or five substituentsindependently selected from the groups as defined herein.

The term “cycloalkyl,” as used herein, refers to a saturated orpartially saturated monocyclic, bicyclic or tricyclic alkyl radicalwherein each cyclic moiety contains from 3 to 12, preferably three toseven, carbon atom ring members. Examples of such cycloalkyl radicalsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and the like.

The terms “heterocyclic” and, interchangeably, “heterocyclyl,” as usedherein, each refer to a saturated, partially unsaturated, or fullyunsaturated monocyclic, bicyclic, or tricyclic heterocyclic radicalcontaining one or more heteroatoms as ring members, wherein each saidheteroatom may be independently selected from the group consisting ofnitrogen, oxygen, and sulfur, and wherein there are typically 3 to 8ring members in each ring. Most commonly heterocyclic rings contain 5 to6 ring members. In some embodiments of this invention heterocyclic ringscontain 1 to 4 heteroatoms; in other embodiments, heterocyclic ringscontain 1 to 2 heteroatoms. In some embodiments, heterocyclic ringscontain heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur or nitrogen and oxygen, or nitrogen, oroxygen.

The term “halo,” or “halogen,” as used herein, refers to fluorine,chlorine, bromine, or iodine. In some embodiments, the halogen may beselected from fluorine, chlorine, or bromine, individually fluorine orchlorine or bromine.

The term “amino,” as used herein, refers to —NRR′, wherein R and R′ areindependently selected from the group consisting of hydrogen, alkenyl,alkoxy, alkoxyalkyl, alkoxycarbonyl, alkyl, alkylcarbonyl, aryl,arylalkenyl, arylalkyl, cycloalkyl, haloalkylcarbonyl, heteroaryl.

The terms “hydroxy” and “hydroxyl,” as used herein, refer to the—OHgroup.

Certain ranges are presented herein with numerical values being precededby the term “about”. The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

This invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

EXAMPLES

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way.

Materials and methods: Sources of the murine lung epithelial (MLE) and293T cells lines are described in Ray et al., Nat. Med., 16(10):1120-1127 (2010), and Chen et al., J. of Biol. Chem.,282(46):33494-33506 (2007). MRC5 cells were from ATCC. Purifiedubiquitin, E1, E2, MG132, Leupeptin, and cycloheximide (CHX) werepurchased from Calbiochem. Mouse monoclonal V5 antibody, the pcDNA3.1Dcloning kit, E. coli Top10 One Shot competent cells, the pENTRDirectional TOPO cloning kits, and Gateway mammalian expression systemwere from Invitrogen. The HECT domain E3 ligase cDNA, scramble shRNA,KIAA0317, PKCξ, and GSK30 shRNA sets were purchased from OpenBiosystems.Nucleofector transfection kits were from Amaxa. Lentiviral packagingsystem and Cobalt beads were from Clontech. Immobilized protein A/Gbeads were from Pierce. In vitro transcription and translation (TnT)kits were from Promega. Cignal SMAD Reporter luciferase Kit (CCS-017L)and mRNA isolation kit were from Qiagen. Complete proteasome inhibitorswere from Roche. KIAA0317 antibodies were from Aviva and Santa Cruz.PIAS and GSK3β antibodies were from Cell Signaling and Santa Cruz. CXCL1and IL6 mouse ELISA kit and TGFβ protein were from R&D Systems. Peptideswere custom synthesized from CHI Scientific. DNA sequencing wasperformed at Genewiz. All small molecule compound analysis was performedby the University of Pittsburgh Mass Spectrometry and NMR facility.

Human Samples: This study was approved by the University of PittsburghInstitutional Review Board. Lung tissues were from the University ofPittsburgh lung transplant tissue bank.

Cell culture: MLE cells were cultured in Dulbecco's Modified EagleMedium-F12 (Gibco) supplemented with 10% fetal bovine serum (DMEM-10).293T cells were cultured in Dulbecco's Modified Eagle Medium (Gibco)supplemented with 10% fetal bovine serum (DMEM-10). MRC5 cells werecultured in Eagle's Minimum Essential Medium (Gibco) supplemented with10% fetal bovine serum (EMEM-10). For protein expression in MLE cells,nucleofection was used following Amaxa's protocol. For proteinoverexpression in 293T cells, Fugene6HD transfection reagents were usedfollowing the manufacturer's protocol. For protein expression in MRC5cells, MRC-5 Cell Avalanche™ Transfection Reagent was used followingmanufacturer's protocol. Cells were treated with TGFβ at 0-2 ng/ml for0-18 h. For FIEL1, PKCξ, or GSK3β knockdown studies in cells, scrambleshRNA, KIAA0317, PKCξ, or GSK3β shRNA were used to transfect cells for48 h. For drug treatment, compounds were solubilized in DMSO beforebeing added to the cells for up to 18 h. Cell lysates were prepared bybrief sonication in 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mMdithiothreitol, 0.025% sodium azide, and 1 mM phenylmethylsulfonylfluoride (Buffer A) at 4° C. For half-life study, MLE cells were exposedto cycloheximide (40 μg/ml) in a time dependent manner for up to 8 h.Cells were then collected and immunoblotted.

Methods

In vitro protein binding assays: PIAS4 protein was immunoprecipitatedfrom 1 mg cell lysate using PIAS4 antibody (goat) and coupled to proteinA/G agarose resin. PIAS4 beads were then incubated with in vitrosynthesized products (50 ul) expressing V5-FIEL1 mutants. After washing,the proteins were eluted and processed for V5-FIEL1 immunoblotting.Similarly, FIEL1 was immunoprecipitated from 1 mg cell lysate usingFIEL1 antibody (rabbit) and coupled to protein A/G agarose resin. FIEL1beads were then incubated with in vitro synthesized products (50 ul)expressing V5-PIAS4 mutants. After washing, the proteins were eluted andprocessed for V5-PIAS4 immunoblotting.

In vitro peptide binding assays: Biotin labeled peptides were firstcoupled to streptavidin agarose beads for 1 h. Beads were then incubatedwith in vitro synthesized FIEL1 or PIAS4 for 18 h. After washing,proteins were eluted and processed for FIEL1 or PIAS4 immunoblotting.

In vitro drug binding assays: FIEL1 protein was HIS-purified from FIEL1expression 293T cells using cobalt beads. Beads were then extensivelywashed prior to exposure to BC-1480 or BC-1485 at differentconcentrations (10⁻⁴ to 100 μM). Purified recombinant PIAS4 protein wasthen incubated with drug-bound FIEL1 beads overnight. Beads were washed,and proteins were eluted and resolved on SDS-PAGE. The relative amountsof PIAS4 detected in the pull-downs were normalized to loading andquantified.

In vitro ubiquitin conjugation assays: The assay was performed in avolume of 25 μl containing 50 mM Tris pH 7.6, 5 mM MgCl₂, 0.6 mM DTT, 2mM ATP, 1.5 ng/μl E1, 10 ng/μl Ubc5, 10 ng/μl Ubc7, 1 μg/μl ubiquitin(Calbiochem), 1 μM ubiquitin aldehyde, and in vitro synthesized V5-PIAS4and FIEL1. Reaction products were processed for V5 immunoblotting.

In vitro kinase assays: The assay was performed in a volume of 25 μlcontaining 50 mM Tris pH 7.6, 100 mM MgCl₂, 0.5 mM ATP, 25 mMβ-Glycerolphosphate, 0.2 μCi γ-32P ATP (Perkin-Elmer), 5 mg/mL BSA,either 500 nM of PKCζ or 500 nM of GSK3β and in vitro synthesizedV5-PIAS4 and V5-FIEL1. Reaction products were processed forautoradiography using Personal Molecular Imager™ (BioRad).

Hydroxyproline Assay: Murine lungs were dried and weighed prior todigestion with HCl. Hydroxyproline concentrations were measuring usingmethods previously described in Kesava Reddy et al., ClinicalBiochemistry, 29(3): 225-229 (1996), and Neuman et al., J. of Biol.Chem., 184(1): 299-306 (1950). Hydroxyproline content was normalized todry lung weight.

SMAD reporter assay: Cignal SMAD Reporter luciferase plasmids wereco-transfected with Empty, FIEL1, PIAS4, CON shRNA, or KIAA0317 shRNAfor 24-48 h before TGFβ treatment for an additional 2-18 h. Cells werethen collected and assayed for firefly and renilla luciferase activity.SMAD transcription activity was normalized by a firefly and renillaluciferase activity ratio.

Immunostaining: MRC5 cells were seeded in 35 mm MatTek glassbottomdishes before the plasmid transfection, inhibitor and TGFβ treatment.Cells were washed with PBS and fixed with 4% paraformaldehyde for 20min, then exposed to 2% BSA, 1:500 mouse α-SMA and goat FN antibodies,and 1:1000 Alexa 488 or Alexa 567 labeled chicken anti-mouse or donkeyanti-goat secondary antibodies sequentially for immunostaining. Nucleuswas counterstained with DAPI. Immunofluorescent cell imaging wasperformed on a Nikon A1 confocal microscope using 405 nm, 488 nm or 567nm wavelengths. All experiments were done with a 60× oil differentialinterference contrast objective lens.

Molecular docking studies and compound design: The docking experimentswere carried out using software from Discovery Studio 3.5. A librarycontaining 500K approved or experimental drugs was first used to screenpotential ligands for FIEL1. FIEL1-HECT domain structural analysisrevealed a major drug binding cavity within the c-terminal of the HECTdomain. The binding cavity was adopted into the LibDock algorithm toscreen for the optimum inhibitor. Based on the docking and best-fitanalysis of suitable ligands, BC-1480 was used as the backbone tosynthesize other compounds.

RT-qPCR, cloning, and mutagenesis: Total RNA was isolated and reversetranscription was performed followed by real-time quantitative PCR withSYBR Green qPCR mixture as described in Butler et al. J. Biol. Chem.2010, 185(9): 6246-6258. All mutant PIAS4 and KIAA0317 plasmidconstructs were generated using PCR-based approaches and appropriateprimers and subcloned into a pcDNA3.1D/V5-HIS vector.

Lentivirus construction: To generate lentivirus encoding FIEL1,Lenti-Plvx-FIEL1 plasmid was co-transfected with Lenti-X HTX packagingplasmids (Clontech) into 293T cells following the manufacturer'sinstructions. 72 h later, virus was collected and concentrated usingLenti-X concentrator.

Animal studies: All procedures were approved by the University ofPittsburgh Institutional Animal Care and Use Committee. For fibrosisstudies, male C57BL/6J mice were deeply anesthetized using aketamine/xylazine mixture, and the larynx was well visualized under afiber optic light source before endotracheal intubation with a 3/40024-gauge plastic catheter. 10⁷ CFU of lentivirus encoding genes forEmpty (E), FIEL1, CON shRNA, or KIAA0317 shRNA was given i.t. for 144 hbefore administration of bleomycin (0.02 U˜0.05 U i.t.) for up to 21days. Animals were euthanized and assayed for BAL protein, cell count,cytokines, and lung infiltrates. Survival studies were performed on micethat were given bleomycin (0.02 U˜0.05 U i.t.). Mice were carefullymonitored over time; moribund, preterminal animals were immediatelyeuthanized and recorded as deceased. For drug studies, mice were deeplyanesthetized as above. Bleomycin (0.05 U i.t.) was given i.t. beforeBC-1480 or BC-1485 (5 mg/kg/d) was administered to the mice throughtheir drinking water. 7-21 d later, animals were euthanized and analyzedas above.

Statistical Analysis: Statistical comparisons were performed withunpaired 2 t-test with p<0.05 indicative of significance. Survival curvewas generated through SPSS (IBM).

Example 1 Compound Synthesis

BC1481 synthesis: 1 mmol BC-1480(4-(2-Oxo-2,3-dihydro-1H-benzoimidazole-5-sulfonylamino) was mixed with1.1 mmol DL-2-Amino-1-(pyrrolidin-1-yl)propan-1-one in 2 ml of DMF. 3mmol of Et3N and 3 mmol of HBTU were then added to the mixture andstirred at RT for 18 h. Products were isolated using chromatography(CHCl3:MeOH 4:1) and dried by vacuum suction to obtain the desiredproduct as a white powder (0.22 g, 52% yield).

BC1485 synthesis: A mixture of BC-14804-(2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-sulfonamido)benzoic acid (66mg. 0.2 mmol), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride (38.3 mg, 0.2 mmol) and 1-Hydroxybenzotriazole hydrate(30.6 mg, 0.2 mmol) in DMF (3 mL) was stirred at room temperature for 10min followed by addition of 2-amino-N,N-dimethylpropanamidehydrochloride (37 mg, 0.24 mmol) and triethylamine (24.3 mg. 0.24 mmol).The reaction was stirred at room temperature under nitrogen overnight.It was concentrated under vacuum. The residue was dissolved indichloromethane (1 mL) and purified by flash chromatography (silica gel,toluene/2-propanol/ammonia hydroxide=80/20/1, v/v/v) to obtain a stickywhite solid. It was suspended in 2N HCl (2 mL), sonicated for 10 min andfiltered. The wet cake was washed with water several times and dried byvacuum suction to obtain the desired product as a white powder (30 mg,35% yield).

BC1486 synthesis: 1 mmol BC-1480(4-(2-Oxo-2,3-dihydro-1H-benzoimidazole-5-sulfonylamino) was mixed with1.1 mmol N1,N1-dimethylvalinamide in 2 ml of DMF. 4 mmol of DIPEA and1.1 mmol of HATU were then added to the mixture and stirred at RT for 18h. Products were isolated using chromatography (CHCl3:MeOH 4:1) anddried by vacuum suction to obtain the desired product as a white powder(0.19 g, 38% yield).

BC1488 synthesis: 1 mmol BC-1480(4-(2-Oxo-2,3-dihydro-1H-benzoimidazole-5-sulfonylamino) was mixed with1.1 mmol 2-Amino-N-benzyl-DL-propanamide in 2 ml of DMF. 4 mmol of DIPEAand 1.1 mmol of HATU were then added to the mixture and stirred at RTfor 18 h. Products were isolated using chromatography (CHCl3:MeOH 6:1)and dried by vacuum suction to obtain the desired product as a yellowpowder (0.35 g, 69% yield).

BC1489 synthesis: 1 mmol BC-1480(4-(2-Oxo-2,3-dihydro-1H-benzoimidazole-5-sulfonylamino) was mixed with1.1 mmol 2-amino-N,N-dimethyl-3-phenylpropanamide in 2 ml of DMF. 4 mmolof DIPEA and 1.1 mmol of HATU were then added to the mixture and stirredat RT for 18 h. Products were isolated using chromatography (CHCl3:MeOH6:1) and dried by vacuum suction to obtain the desired product as ayellow powder (0.25 g, 51% yield).

DL-2-Amino-1-(pyrrolidin-1-yl)propan-1-one: 1 mmol BC-1480(4-(2-Oxo-2,3-dihydro-1H-benzoimidazole-5-sulfonylamino) was mixed with1.05 mmol alaninamide in 2 ml of DMF. 3 mmol of DIPEA and 1.05 mmol ofHATU were then added to the mixture and stirred at RT for 18 h. Productswere isolated using chromatography (CHCl3:MeOH 4:1). Yield (0.24 g,55%). High resolution ESI-MS: 432.1340. Calculated for C19H22O5N5S+[M+H+].

Example 2 FIEL1-PIAS4 Pathway in Pulmonary Fibrosis

Ubiquitin was overexpressed, which decreases PIAS4 t_(1/2) from ˜6 h to2 h. The degradation of PIAS4 occurs in a ubiquitin-dependent (FIG. 9A).Specifically, this occurs through the proteasome as addition of theproteasomal inhibitor MG132 to cells significantly increased PIAS4protein half-life, an effect unobserved with the addition of thelysosomal inhibitor Leupeptin (FIG. 9B).

E3 ligases potentially involved in PIAS4 ubiquitination were screened.It was determined that the HECT domain E3 ligase KIAA0317 regulatesPIAS4 protein stability (data not shown). Interestingly, KIAA0317encodes two major isoforms, 823aa and 789aa and a previous studyrevealed that the longer form (823aa, termed AREL1) of KIAA0317regulates the ubiquitination of the apoptosis proteins SMAC, HtrA2, andARTS, as described in Kim et al., J. Biol. Chem., 288(17): 12014-12021(2013). However, it is found that the shorter isoforms (789aa, termedFIEL1, Fibrosis Inducing E3 Ligase 1) behave distinctly in cells. First,only overexpression of the shorter isoform of KL140317 (FIEL1 )decreased PIAS4 protein levels (FIG. 1A). Compared to AREL1, FIEL1interacted with much more PIAS4 protein in an in vitro pull down assay(FIG. 1B). Moreover, overexpression of FIEL1 in murine lung epithelialcells (MLE), HeLa and 293T co-migrated with the endogenous protein,which suggested that FIEL1 is the predominant KIAA0317 isoform in all ofthese cell lines (FIG. 9C). FIEL1 regulates PIAS4 ubiquitination invitro (FIG. 1C). FIEL1 expression selectively decreased PIAS4, overother family members, in a dose-dependent manner in MLE cells (FIG. 9D).A randomly selected HECT E3 ligase, UBE3B, was also tested as a negativecontrol (FIG. 9D). FIEL1 expression in HeLa and 293T cells alsodecreased PIAS4 protein levels (FIG. 9E). Conditional expression ofFIEL1 in MLE cells using a doxycycline-inducible plasmid resulted inPIAS4 protein degradation in a doxycycline dose-dependent manner (FIG.9F). Further, FIEL1 expression dramatically decreased PIAS4 proteinlevels, whereas FIEL1 knockdown using shRNA stabilized PIAS4 (FIGS.1D-E) but had no effect on PIAS4 mRNA levels (data not shown). TGFβtreatment increased FIEL1 protein and decreased PIAS4 protein in both adose and time-dependent manner in human primacy lung fibroblast MRC5cells (FIG. 1F). TGFβ treatment also increased the association of PIAS4and FIEL1 (FIG. 1F). Last, TGFβ treatment drastically prolonged FIEL1protein t_(1/2), and also decreased PIAS4 protein t_(1/2) (FIG. 9G).

To identify the ubiquitin acceptor site within PIAS4, a candidatemapping approach was used in which PIAS4 lysine mutants were constructedand tested using various assays (FIG. 10A). The PIAS4 K31R mutantexhibited an extended t_(1/2) compared to wild-type PIAS4 (FIG. 10B). Ofseveral PIAS4 point mutants tested, only PIAS4 K31R exhibited partialresistance to ubiquitin degradation (FIG. 10C). To confirm thesignificance of the FIEL1-PIAS4 pathway in vivo, FIEL1 and PIAS4 proteinlevels in lung tissues from five control subjects and five subjects withIPF were assayed. Subjects with IPF had significantly less PIAS4 proteinand more immunoreactive FIEL1 protein in their lungs versus controlsubjects (FIG. 1G). This pathway was also tested in bleomycin inducedmurine lung fibrosis, as described in Tager et al., Nat. Med.,14(1):45-54 (2008); and Jiang et al., J. Clinical Investigation,120(6):2049-2057 (2010). Bleomycin challenge significantly increasedFIEL1 protein levels and decreased PIAS4 protein levels in mice lungswith a maximum effect at day 14 (FIG. 1H). These results suggest thatthe FIEL1-PIAS4 pathway is functional and important in individuals withIPF.

Example 3 FIEL1 Promotes TGFβ Signaling

FIEL1 promoting TGFβ signaling was investigated by measuring SMADtranscriptional activity using a SMAD reporter assay (Qiagen). FIEL1overexpression increased SMAD driven luciferase activity upon TGFβstimulation in a dose dependent manner (FIG. 2A). By decreasing PIAS4protein, expression of FIEL1 also decreased SMAD3 sumoylation (FIG. 2B)and further promoted SMAD3 nuclear import (FIG. 2C). Upon FIEL1expression, fibrotic markers Fibronectin (FN) and alpha smooth muscleactin (α-SMA) in MRC5 cells were measured. FIEL1 expression increasedthe expression of FN and α-SMA (FIG. 2D). This was also confirmed by FNand α-SMA immunostaining in MRC5 cells (FIG. 2E). When FIEL1 isknockdown using several shRNAs, a significantly reduced SMAD drivenluciferase activity was observed (FIG. 2F). FIEL1 knockdown increasedPIAS4 protein, increased SMAD3 sumoylation (FIG. 2G), and furtherdecreased SMAD3 nuclear import (FIG. 2H). FIEL1 knockdown also reducedFN and α-SMA expression in MRC5 cells upon TGFβ treatment (FIGS. 2I, J).

Example 4 PIAS4 Phosphorylation by PKCξ is Required for FIEL1 Binding

The FIEL1 binding site within PIAS4 was investigated. Mapping studieswere by constructing several deletional mutants of PIAS4 and cloningthem into a pcDNA3.1D/V5-HIS vector (FIG. S3A). It was determined thatFIEL1 binds within the N-terminal 25 amino acids of PIAS4 (FIG. S3B,lower blot). An alanine scan study within this region suggested thatboth S18 and Q21 are important for FIEL1 interaction, as both S 18A andQ21A mutants drastically lost binding with FIEL1 (FIG. S3C). A kinasescreen was performed and it was determined that PKCξ interacts withPIAS4 via Co-IP (FIG. 3A). Erk1 and PKCα were also included asspecificity controls. PKCξ expression also decreased PIAS4 protein levelin a dose-dependent manner, whereas JNK1 expression was not able toachieve such an effect (FIG. 3B). Moreover, PKCξ knockdown using shRNAdrastically stabilized PIAS4 protein in a t_(1/2) study (FIG. 3C),whereas PKCξ expression decreased PIAS4 t_(1/2) to ˜2 h (FIG. 3D).

To confirm that PIAS4 is phosphorylated in vitro, cells were lysed andsubjected to PIAS4 IP, and using phospho-serine antibodies, a band wasdetected which migrated to the predicted size of PIAS4 (FIGS. 3E-F, ˜60kDa). TGFβ stimulation also drastically increased PIAS4 serinephosphorylation and PKCξ binding, but not PKCα binding (FIGS. 3E-F). Itwas observed that PKCξ directly phosphorylated PIAS4 in an in vitrokinase assay (FIG. 3G). PKCξ knockdown also protected PIAS4 fromphosphorylation and degradation during TGFβ treatment (FIG. 3H).Compared to WT PIAS4, S18A and S14/S18A double mutant exhibited adramatic decrease in phosphorylation and offered significant resistanceto degradation during TGFβ treatment (FIG. 3I). PIAS4 S18A, Q21A, andS18/Q21A double mutants also exhibited much longer half-lives (FIG. S3D)and resisted degradation from FIEL1 co-expression (FIG. S3E). Last, apeptide binding experiment was performed in which four biotin-labeledsynthetic peptides were bound to streptavidin-agarose beads and servedas bait for FIEL1 binding (FIG. 3J). The peptide with S18phosphorylation (P2) showed the strongest binding to FIEL1; the peptidewith Q21 mutation (P3) offered drastically decreased FIEL1 interaction(FIG. 3J). These experiments suggested that PKCξ is an authenticregulator of PIAS4 protein stability; Q21 and phosphorylated S18 ofPIAS4 are both required for FIEL1 interaction.

Example 5 GSK3β Phosphorylation of FIEL1 is Required for PIAS4 Targeting

The PIAS4 binding site within FIEL1 was investigated. A similar mappingstudy as above was performed by constructing several deletional mutantsof FIEL1 and cloning them into a pcDNA3.1D/V5-HIS vector (FIG. 11A). Amapping study was done similarly to FIG. 11 using a PIAS4 antibody toconstruct PIAS4 beads as the bait. It was first determined that PIAS4binds within the last 189 residues of the C-terminal of FIEL1 (FIGS.12B-C). Additional mapping suggested that PIAS4 interacts with the last10 amino acids of FIEL1 (FIGS. 12D-E). An alanine scan study within thisregion suggested that both P779 and T783 are important for PIAS4interaction, as both P779A and T783A mutants drastically lost bindingwith PIAS4 (FIG. 12F). Compared with WT FIEL1, neither P779A nor T783Amutant expression decreased PIAS4 protein levels (FIG. 12G). It was alsodetermined that C770 is a potential active site of FIEL1 as the C770Smutant also failed to decrease PIAS4 protein level (FIG. 12G). SNPdatabase analysis indicated a naturally occurring polymorphism(rs371610162) within FIEL1 (P779L). This mutation was further tested ina binding assay and showed that T783A, P779L, and P779L/T783A doublemutant all lost interaction with PIAS4 (FIG. 12H).

A kinase screen was performed and it was determined that GSK3β interactswith FIEL1 via Co-IP (FIG. 4A). JNK2 and PKCα were also included asspecificity controls. WT GSK3β overexpression decreased PIAS4 proteinlevels in a dose dependent manner, and PIAS4 protein levels decreasedmore dramatically when cells was transfected with a constitutivelyactivated GSK3β hyper mutant plasmid (FIG. 4B). Moreover, a CHX t_(1/2)study suggested that WT GSK3β ectopic expression decreased PIAS4 t_(1/2)to ˜4 h, whereas the more potent GSK3β hyper mutant further decreasedPIAS4 t_(1/2) to 2 h (FIG. 4C). TGFβ stimulation drastically increasedFIEL1 threonine phosphorylation (FIG. 4D), and GSK3β knockdowndrastically stabilized PIAS4 in a t_(1/2) study (FIG. S5A). Moreover,GSK3β knockdown also protected FIEL1 from threonine phosphorylation andPIAS4 degradation with TGFβ treatment (FIG. 4E).

Using the lung lysates from FIG. 1F, a Co-IP experiment was performedwhere FIEL1 protein was immunoprecipitated and immunobloted for PIAS4and p-Thr. a positive association between PIAS4 and p-Thr signal wasobserved, which suggested that FIEL1 threonine phosphorylation isessential for PIAS4 binding (FIG. 4F). The role of FIEL1 T783 inregulating PIAS4 protein stability was further studied. FIEL1 T783Amutant overexpression completely failed to decrease PIAS4 proteinlevels. However, phosphorylation mimic T783D mutant FIEL1 expressiondecreased PIAS4 protein levels more dramatically compared to WT FIEL1expression (FIG. 13B). Moreover, a t_(1/2) study suggested that WT FIEL1expression decreased PIAS4 t_(1/2) to ˜4 h, whereas the more potentphosphorylation mimic T783D further decreased PIAS4 t_(1/2) to ˜2 h(FIG. 13C). The FIEL1 T783A mutant was resistant to GSK3βphosphorylation in an in vitro kinase assay (FIG. 4G). The FIEL1 T783Amutant also exhibited drastically decreased phosphorylation in cells(FIG. 4H). A peptide binding experiment was also performed where fourbiotin-labeled synthetic peptides were bound to streptavidin-agarosebeads and served as bait for PIAS4 binding (FIG. 4I). Peptide withphosphorylation at T783 (P2) showed the strongest binding to PIAS4;peptide with no phosphorylation (P1) or T783A mutant (P4) offereddrastically decreased PIAS4 interaction. FIEL1 T783A and P779Lexpression both failed to decrease PIAS4 protein levels (FIG. 13D) orhalf-lives (FIG. S5E). FIEL1 T783A/P779L double mutant expression showeda dominant negative phenotype by increasing PIAS4 protein levels (FIG.13D). PIAS4 peptide 2 was used with S18 phosphorylation as bait in an invitro binding assay (FIG. 4J). Both P779L and T783A mutants drasticallylost binding with PIAS4, which is similar to FIG. S4H. Similarly, FIEL1Peptide 2 with phosphorylation at T783 (P2) was used to reconfirm theimportance of S18 phosphorylation and Q21 within PIAS4 for FIEL1interaction (FIG. 4K), which is similar to FIG. S3C. Last, FIEL1T783A/P779L dominant negative double mutant overexpression protectedPIAS4 from TGFβ treatment (FIG. 13F). These experiments suggested thatthe GSK3β phosphorylation of FIEL1 is required for PIAS4 targeting, andFIEL1 residues P779 and phosphorylated T783 are both required for PIAS4interaction.

Example 6 Gene Transfer of FIEL1 Exacerbates Bleomycin Induced LungInjury In Vivo

This example considers whether expression of FIEL1 in vivo alters hostinflammatory responses and induce fibrotic lung injury. To extend theabove observations in vivo, mice were infected with an empty lentivirusor lentivirus encoding FIEL1 for 144 h (10⁷ cfu/mouse, intratracheally[i.t.]). Mice were then challenged with bleomycin (0.02 U i.t.) for anadditional 1-21 days. Mice were euthanized to analyze parameters offibrotic lung injury.

Bleomycin has been wildly used to study IPF in murine models. As shownin FIG. 5A, the increased BAL total protein concentration that occursafter bleomycin injury in control mice was significantly increased inmice overexpressing FIEL1. Chemokine CXCL1 levels in BALs were alsosignificantly increased in mice overexpressing FIEL1 (FIG. 5B). Asimilar increase in IL-6 levels in BALs from mice overexpressing FIEL1was also observed (data not shown). The role of FIEL1 in the lung'sinflammatory response to bleomycin was investigated (FIGS. 5C-F). Amarked increase in total inflammatory cells in the BAL fluid from miceoverexpressing FIEL1 was observed (FIG. 5C). Specifically, thedifferential cell counts of the BALs revealed that the total increase ininflammatory cells was mostly due to neutrophils and lymphocytes, withthe exception of macrophages on day 7 (FIGS. 5D-F). FIEL1 expression inmice also significantly reduced survival (FIG. 5G). A marked increase inlung fibrosis in mice overexpressing FIEL1 as demonstrated bysignificantly increased hydroxyproline content was observed (FIG. 5H).Bleomycin challenge also showed changes consistent with peribronchiolarand parenchymal fibrosis in a time-dependent manner (FIGS. 5I, H&E). Theextent of these changes present in FIEL1 expression mice wassubstantially increased as compared to the empty control (FIGS. 5I,H&E). Elevated lung collagen visualized by Mason Trichrome staining alsosuggested that FIEL1 expression exacerbates bleomycin-induced lunginjury (FIG. 5I, Trichrome).

Example 7 FIEL1 Knockdown Ameliorates Bleomycin-Induced Lung Injury InVivo

To further confirm the role of FIEL1 in lung fibrosis and inflammation,in vivo knockdown studies were pursued. Mice were first infected withlentivirus encoding CON shRNA or KIAA0317 shRNA for 144 h (10⁷CFU/mouse,i.t) and then challenged with bleomycin (0.05 U i.t.) for an additional1-21 days.

FIEL1 knockdown significantly decreased BAL protein concentrations andChemokine CXCL1 levels (FIGS. 6A, B). FIEL1 knockdown also significantlydecreased BAL total cell counts (FIG. 6C). Specifically, thedifferential cell counts of BALs revealed that the total decrease ininflammatory cells was mostly due to neutrophils and lymphocytes, withthe exception of macrophages on day 3 and 21 (FIGS. 6D-F). FIEL1knockdown in mice also significantly improved survival (FIG. 6G). Amarked decrease in lung fibrosis in FIEL1 knockdown mice as demonstratedby a significant decrease in hydroxyproline content was observed (FIG.6H). Peribronchiolar and parenchymal fibrosis were also substantiallydecreased in FIEL1 knockdown mice (FIGS. 6I, H&E). Decreased lungcollagen visualized by Mason Trichrome staining also suggested thatFIEL1 knockdown ameliorates bleomycin-induced lung injury (FIG. 6I,Trichrome).

Example 8 FIEL1 Domain Analysis and Inhibitor Design

This example tests that small molecule inhibition of the HECT domainwould disrupt FIEL1 targeting its substrate, PIAS4, because FIEL1harbors a conserved HECT domain within its C-terminus which carries outthe E3 ligase activity of transferring ubiquitin to the substrate. Ahomology model using the NEDD4 HECT domain was first constructed(2XBF.pdb) (FIG. 7A). NEDD4 HECT domain was described in Umadevi N etal. Acta crystallographica Section F, Structural biology andcrystallization communications, 61(Pt 12): 1084-1086 (2005), andKamadurai HB et al. Molecular cell. 36(6): 1095-1102 (2009). A majorcavity within the C-terminal of the FIEL1 HECT domain was observed,which is also required for PIAS4 binding (FIG. 12). Using moleculardocking analysis and score-ranking operations on the predictedFIEL1-HECT domain 3-D structure model, potential ligands that might fitthe HECT domain cavity was assessed (FIG. 7A).

These docking experiments were conducted using the LibDock program fromDiscovery Studio 3.5, and a library containing 500 k small moleculecompounds was first used to screen potential ligands for the FIEL1-HECTdomain. 19 compounds were selective for initial round of testing. 9/19compounds showed good activity (IC50<1 uM) blocking FIEL1 in MLE cellsindicated by increase substrate PIAS4 protein (FIG. 14). BC-1480(4-(2-Oxo-2,3-dihydro-1H-benzoimidazole-5-sulfonylamino)-benzoic acid)was further selected as a backbone to develop small-molecule inhibitors(FIG. 7B). BC-1485 was synthesized by reacting alaninamide with BC-1480(FIG. 15A). As shown in FIGS. S7B-C, BC-1485 fits in the HECT domaincavity fairly well having several electrostatic interactions withGLN774, HIS 788, ILE 776 and THR783 (FIGS. 15B-C). Compared to BC-1480,BC-1485 exhibited >100 fold activity in disrupting the FIEL1/PIAS4interaction (FIG. 7C) and increasing PIAS4 protein levels (FIG. 7D).Indeed, BC-1485 decreased the expression of α-SMA in MRC5 cells withIC₅₀≈32 nM, whereas BC-1480 decreased the expression of α-SMA withIC₅₀≈4 μM (FIG. 15D). This was also confirmed by FN and α-SMAimmunostaining in MRC5 cells (FIG. 15E). BC-1485 also stabilized PIAS4by extending its half-life (FIG. 15F). Last, BC-1485 did notsignificantly alter KIAA0317 or PIAS4 mRNA levels (FIG. 15G).

To further assess the anti-fibrotic activity of BC-1485, it was testedin vivo using a bleomycin model. Mice were first challenged withbleomycin (0.05 U i.t.) for up to 21 days. BC-1485 was given in drinkingwater with an estimated dose of 5 mg/kg/d. BC-1485 significantlydecreased BAL protein concentrations, CXCL1, and IL-6 levels (FIGS. 7E,F, FIG. 16A). BC-1485 also significantly decreased BAL total cell counts(FIG. 7G). Specifically, the differential cell counts of BAL cellsrevealed that the total decrease in inflammatory cells was mostly due toneutrophils and lymphocytes, with the exception of macrophages on day 7and 21 (FIGS. 16B-D). BC-1485 also significantly improved survival (FIG.7H). A marked decrease in lung fibrosis in mice treated with the FIEL1inhibitor BC-1485 as demonstrated by a significant decrease inhydroxyproline content was observed (FIG. 7I). Peribronchiolar andparenchymal fibrosis were also substantially decreased by BC-1485 (FIG.16E). Decreased lung collagen visualized by Mason Trichrome stainingalso suggested that BC-1485 ameliorates bleomycin-induced lung injury(FIG. 7J). Hence, small-molecule targeting of the FIEL1/PIAS4 pathwayreduced the severity of fibrosis in a preclinical model.

Example 9 BC1485 Inhibits JAK/Stat Pathway

HCT8 cells were pretreated with BC-1485 at different concentrations for16 h before exposed to DSS (2%, 16 h) or IFNγ (10 ng/ml, 1 h). Cellswere collected and assayed for protein immunoblotting. BC1485effectively decreases JAK1 and JAK2, p-Stat1 and p-Stat3 protein levels,and thus inhibits the signaling transduction in JAK/STAT pathway.

Example 10 BC1485 Reduces Dextran Sulfate Sodium (DSS) Induced AcuteColonic Inflammation

C57BL6 mice were fed with water ad lib containing 2.5% dextran sulfatesodium (DSS) for up to 7 days. Mice were treated with either vehicle(control) or BC-1261 (administered into drinking water at 30 ug/ml, ˜5mg/kg/d dosing). Mice were monitored daily, weights were measured andgraphed, shown in FIG. 20A. Mice were then euthanized, the length of thecolon and weight of the spleen were measured and graphed, shown in FIGS.20B-D. Colonic tissues were also analysed for TNFα, IL1 and IL6 shown inFIGS. 20E H&E staining was performed on colonic sections. The data inFIG. 20F represent n=6 mice/group, *P<0.05 versus DSS and **P<0.05versus control. The results shows that BC1485 effectively reducesdextran sulfate sodium induced acute colonic inflammation in mice.

What is claimed is:
 1. A pharmaceutical formulation comprising acompound represented by Formula (I):

wherein: R is

and wherein: W is selected from the group consisting of H, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedaryl, optionally substituted cycloalkyl, optionally substitutedheterocyclic, halogen, amino, and hydroxy; X is selected from the groupconsisting of H, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted heterocyclic, halogen, amino, and hydroxy; Y isselected from the group consisting of H, optionally substituted alkyl,optionally substituted aryl, optionally substituted cycloalkyl, andoptionally substituted heterocyclic; Z is selected from the groupconsisting of H, optionally substituted alkyl, optionally substitutedaryl, optionally substituted cycloalkyl, and optionally substitutedheterocyclic; and wherein Y and Z optionally bind together to form aring; R′ is selected from the group consisting of H, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcycloalkyl, and optionally substituted heterocyclic; R″ is independentlyselected from the group consisting of H, optionally substituted alkyl,optionally substituted aryl, optionally substituted cycloalkyl, andoptionally substituted heterocyclic; wherein one or more of the alkyl,cycloalkyl, heterocycloalkyl, aryl or heteroaryl may be substituted byone or more C₁ -C₆ alkoxy, halogen or deuterium; or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable excipient. 2.The pharmaceutical formulation of claim 1, wherein: (a) W is selectedfrom the group consisting of H, optionally substituted alkyl, andoptionally substituted aryl; (b) X is selected from the group consistingof H, optionally substituted alkyl, and optionally substituted aryl, and(c) W and X cannot both be H.
 3. The pharmaceutical formulation of claim1, wherein: (a) Y is selected from the group consisting of H, optionallysubstituted alkyl, and optionally substituted aryl, (b) Z is selectedfrom the group consisting of H, optionally substituted alkyl, andoptionally substituted aryl, and (c) Y and Z cannot both be H.
 4. Thepharmaceutical formulation of claim 1, wherein R′ is H.
 5. Thepharmaceutical formulation of claim 1, wherein R″ is H.
 6. Thepharmaceutical formulation of claim 1, wherein R is


7. The pharmaceutical formulation of claim 1, wherein R is


8. The pharmaceutical formulation of claim 1, wherein R is


9. The pharmaceutical formulation of claim 1, wherein R is